Protozoan parasites that belong to the genus Leishmania from the Trypanosomadae family, causes Leishmaniasis in humans, which affects 350 million people in 88 countries worldwide. The parasites are principally transmitted to humans following the bite of female phlebotomine sandflies, which carry the infectious stage of the organism. Leishmaniasis comprise three major clinical syndromes principally determined by the species of infecting parasites, which vary geographically. Few therapies are currently available, but strong side effects and parasite resistance limit their effectiveness. The recent advancements in determining several trypanosomatid genomes provided an invaluable tool to gather information regarding the molecular and biochemical mechanisms used by these organisms. However, it has proven difficult to correctly assign specific functions to gene coding sequences because the sequence homology between these parasites and their higher eukaryotic counterparts is extremely low. Until recently, little effort was invested in the Leishmania cap-binding complexes in the human stage, which are required to initiate protein synthesis in parasites. In this proposal, we focus on three translation initiation factors from Leishmania major in the human stage: LeishIF4E-1, Leish4E-IP and LeishIF3. While LeishIF4E-1 has emerged as the crucial cap-binding protein, it is hypothesized that Leish4E-IP and LeishIF3 are, respectively, a regulator of translation initiation and a scaffold protein responsibl for recruiting the small ribosomal subunit at the 5' end of messenger RNAs. To investigate whether this is the case, we will apply a combination of biophysical techniques, including NMR spectroscopy and X-ray crystallography, and molecular biology approaches to study the structure/function of these factors in vitro and within parasites. Once we determine indispensable interactions that promote translation initiation in the parasite, we will explore these potential targets in high-throughput (HTP) small molecule inhibitor screens. This proposal will allow us to highlight similarities and differences between translation initiation in human cels and Leishmania parasites, and validate the Leishmania translation machinery as a potential drug target. This concept is backed by data obtained previously in the Wagner lab where a crucial interaction between two human translation initiation factors (eIF4E/eIF4G) has been targeted in a HTP screen and led to the discovery of compounds with anti-cancer activity. The research will pursue three specific aims: 1. Determine the structure of the LeishIF4E-1 complex with its ligand cap-4 and Leish4E-IP. 2. Characterize the LeishIF3 recruitment to the Leishmania cap-binding complex. 3. Develop assays for HTP screens in search of Leishmania inhibitory compounds.